Ionization behavior of native and mutant insulins: pK perturbation of B13-Glu in aggregated species.
Upscale titration from pH 2.5 to 11.2 is used as a means for probing solvent accessibility of ionizing groups in zinc-free preparations of native and mutant insulins. Stoichiometry and pK alpha values of ionizing groups in the titration curves are determined by iterative curve fitting. Under denaturing conditions, the titration curve of human insulin is in good agreement with that predicted from the sum of unperturbed titrations of the constituent ionizing groups and yields an apparent isoionic point of 5.3. Under nondenaturing conditions where aggregation and precipitation occur, titrations show that only five out of six carboxylate residues of human insulin ionize in the expected region. Consequently, one carboxylate ionization is masked and the apparent isoionic point located at pH 6.4. Correlation between ionization behavior and patterns of aggregation and solubility is established by titrations of mutant insulins and of dilute native insulin. Titration of an unusually soluble species, B25-Phe----His, shows that precipitation is not responsible for the masked carboxylate ionization of native insulin. Titrations of mutants B13-Glu----Gln and B9-Ser----Asp show that the masked ionization probably originates from monomer-monomer interactions in the insulin dimer. We conclude that the B13-Glu side chain is responsible for the masked carboxylate ionization in aggregated forms of human insulin.
Kaarsholm
N C
NC
Novo Research Institute, Bagsvaerd, Denmark.
Havelund
S
S
Hougaard
P
P
D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011199:Potentiometry; D011487:Protein Conformation
D007328:Insulin; D046911:Macromolecular Substances